A complete understanding of the molecular mechanisms of disease requires elucidation of gene regulatory mechanisms. This proposal focuses upon translation-level gene control mechanisms and the experiments are directed at the biochemistry and structure/function relationships of ribonucleic acids (RNA) and specific RNA-protein interactions. The goals of this project are to define the molecular basis of selective messenger RNA (mRNA) translation and to characterize determinants present in RNAs and proteins that underlie formation of specific ribonucleoprotein complexes. The model system for study centers upon alfalfa mosaic virus (AlMV), the gene arrangement and protein sequences of which show similarities to the human alphaviruses that cause severe encephalitis. AlMV RNA 4, the viral coat protein messenger RNA, is an efficiently-translated template, and the translated coat protein binds specifically to the 3' untranslated region of its messenger RNA. This RNA-protein interaction is required both for virus assembly and for virus replication; moreover, progress to date suggests it may also be important for its efficient translation. Deletion of the 3' untranslated region of AlMV RNA 4, which includes the coat protein binding domain, severely compromises mRNA translational efficiency. Control experiments show that diminished translation is not due to destabilization of the mRNAs. A 39-nucleotide minimal coat protein binding site has been identified at the 3' terminus of AlMV RNA 4, and it has been demonstrated that the amino-terminus of the coat protein is both necessary and sufficient for binding RNA. The functional significance of the amino-terminal RNA binding domain was confirmed by showing that coat protein peptides (25 or 38 amino acids in length) bind RNA specifically, alter RNA conformation, and activate the initial steps of virus replication. The changes in RNA conformation observed upon peptide binding are very similar to conformational changes observed when peptides from the Tat and Rev proteins of human immunodeficiency virus (HIV) bind their RNA targets. The specific aims for the continuation period include characterizing the role of the AlMV RNA 43' untranslated region in facilitating translation. The minimal 3' sequence or structure that will maintain efficient translation will be mapped by deletion analysis; moreover, the effect of coat protein binding on RNA translational efficiency will be tested. These experiments couple well with efforts aimed at a biochemical and biophysical analysis of peptide binding to AlMV RNA 4 fragments. A combination of random peptide libraries and in vitro selection of RNA ligands will be used to precisely define RNA and protein determinants, and the structure of the RNA and RNA-protein complexes will be probed by chemical interference studies and hydroxyl radical footprinting. Functional analysis of the complexes is tested by vines replication assays in plant protoplasts. The biochemical studies are sufficiently advanced that we have initiated structural analysis of the peptide-RNA complex by NMR spectroscopy and by attempting to grow co- crystals of peptide bound to RNA.